Specific analogues uncouple transport, signalling, oligo-ubiquitination and endocytosis in the yeast Gap1 amino acid transceptor

Abstract

The Saccharomyces cerevisiae amino acid transceptor Gap1 functions as receptor for signalling to the PKA pathway and concomitantly undergoes substrate-induced oligo-ubiquitination and endocytosis. We have identified specific amino acids and analogues that uncouple to certain extent signalling, transport, oligo-ubiquitination and endocytosis. L-lysine, L-histidine and L-tryptophan are transported by Gap1 but do not trigger signalling. Unlike L-histidine, L-lysine triggers Gap1 oligo-ubiquitination without substantial induction of endocytosis. Two transported, non-metabolizable signalling agonists, β-alanine and D-histidine, are strong and weak inducers of Gap1 endocytosis, respectively, but both causing Gap1 oligo-ubiquitination. The non-signalling agonist, non-transported competitive inhibitor of Gap1 transport, L-Asp-γ-L-Phe, induces oligo-ubiquitination but no discernible endocytosis. The Km of L-citrulline transport is much lower than the threshold concentration for signalling and endocytosis. These results show that molecules can be transported without triggering signalling or substantial endocytosis, and that oligo-ubiquitination and endocytosis do not require signalling nor metabolism. Oligo-ubiquitination is required, but apparently not sufficient to trigger endocytosis. In addition, we demonstrate intracellular cross-induction of endocytosis of transport-defective Gap1(Y395C) by ubiquitination- and endocytosis-deficient Gap1(K9R,K16R). Our results support the concept that different substrates bind to partially overlapping binding sites in the same general substrate-binding pocket of Gap1, triggering divergent conformations, resulting in different conformation-induced downstream processes.

Fig 1

Identification of transported, partially or largely competitive inhibitors without signalling capacity.A. Activation of the PKA target trehalase in the wild-type strain after addition of 5 mM l-citrulline (•), l-histidine (○), l-lysine (○) or l-tryptophan (△) to nitrogen-starved cells.B. Gap1-dependent uptake. Transport of 5 mM l-citrulline, l-histidine, l-lysine or l-tryptophan in wild-type (black bars) and gap1Δ (white bars) strains.C. The three non-signalling amino acids are very poor nitrogen sources. Growth on 5 mM l-citrulline (•, ○), l-histidine (○, △), l-lysine (♦, ⋄), l-tryptophan (▾, ▿) or l-asparagine (▼, ▽) in wild-type (closed symbols) and gap1Δ (open symbols) strains.D. l-histidine, l-lysine and l-tryptophan act as inhibitors of Gap1 transport. Transport of 1 mM l-citrulline measured in the presence of different concentrations l-histidine, l-lysine and l-tryptophan (0, 0.5, 1, 5 and 10 mM, white bars to black bars).E. l-histidine, l-lysine and l-tryptophan act as partially or largely competitive inhibitors of Gap1 transport. Transport of five concentrations (0.5, 1, 2.5, 5 and 10 mM, white bars to black bars) of l-citrulline measured without inhibitor or in the presence of 0.125 mM l-histidine, 0.5 mM l-lysine or 0.125 mM l-tryptophan. These values are also shown as a Lineweaver-Burk plot (inset): no inhibitor (•), or 0.125 mM l-histidine (○), 0.5 mM l-lysine (○), or 0.125 mM l-tryptophan (△).F. Transport of the non-signalling amino acids is reduced by mutagenesis of Ser³⁸⁸ or Val³⁸⁹ to cysteine. Transport of 5 mM l-citrulline, l-histidine, l-lysine or l-tryptophan by a wild-type (1), gap1^S388C (2, 3) and a gap1^V389C (4, 5) strain, without (2, 4) or with (3, 5) pre-addition of 10 mM MTSEA.Error bars in (A) to (F) represent standard deviation (s.d.) between biological repeats.

Fig 2

All three non-signalling amino acids act as partially or largely competitive inhibitors of l-citrulline induced trehalase activation.A–C. Activation of the PKA target trehalase in nitrogen-starved cells of the wild-type strain after addition of (A) 5 mM l-citrulline in the presence of 0 mM (•), 2 mM (○), 5 mM (○), 10 mM (△) or 20 mM (○) l-histidine; (B) 2 mM l-citrulline in the presence of 0 mM (•), 10 mM (○), 20 mM (○), 50 mM (△) or 100 mM (○) l-lysine; (C) 5 mM l-citrulline in the presence of 0 mM (•), 1 mM (○), 2 mM (○), 5 mM (△) or 10 mM (○) l-tryptophan.D. Activity of trehalase was measured 20 min after addition of the indicated l-citrulline concentrations in the absence or presence of 1 mM l-histidine, 10 mM l-lysine or 1 mM l-tryptophan. These values are also shown as a Lineweaver-Burk plot (inset): no inhibitor (•), 1 mM l-histidine (○), 10 mM l-lysine (○) or 1 mM l- tryptophan (△). Error bars represent s.d. between biological repeats.

Fig 3

The transported non-signalling amino acid l-lysine does not trigger substantial endocytosis but triggers Gap1 oligo-ubiquitination, and counteracts l-citrulline induced internalization.A. Gap1-GFP localization in wild-type cells is shown 60, 120 and 180 min after addition of 5 mM of l-citrulline or the non-signalling amino acids l-histidine or l-lysine, to nitrogen-starved cells (nitrogen starvation medium, NSM).B. Gap1-GFP localization in wild-type cells is shown before and 60 min after addition of 5 mM l-citrulline, either without (+0 mM l-lysine), or together with different concentrations of l-lysine (10, 20, 50 or 100 mM) to nitrogen-starved cells.C. Analysis of Gap1-GFP stability in membrane-enriched (P13) fractions at different time points (0, 30, 60, 120 and 180 min) after addition of l-citrulline, l-histidine or l-lysine to nitrogen-starved cells. Western blot was carried out with HRP-anti-GFP antibody, showing levels of Gap1-GFP (10 s exposure), or free GFP at 60 s of exposure of the same blot. Normalization of the loading is shown with anti-Pma1 antibody. Luminescent arbitrary units (LAU) × 10⁻⁶ are shown as ratio between the Gap1-GFP band and Pma1 band for each time point.D. Analysis of Gap1 ubiquitination status in nitrogen-starved cells expressing endogenous GAP1 and induced with 10 μM CuSO₄ for 30 min prior to addition of nitrogen source, for moderate overexpression (OE) of myc-ubiquitin from the P_CUP1-myc-Ubi URA3 plasmid, pMRT7. P13 fractions were collected at different time points (0, 30, 60, 120 and 180 min) after addition of 5 mM l-citrulline, l-histidine or l-lysine to nitrogen-starved cells. Upper panels: Western blot with anti-Gap1 antibody. Bottom panels: Western blot with anti-Pma1 antibody as loading control. Luminescent arbitrary units (LAU) × 10⁻⁶ are shown as ratio between the Gap1 band and Pma1 band for each time point to assess relative disappearance of the Gap1 band, consistent with endocytosis. The ratios between di- or tri-ubiquitinated to non-ubiquitinated Gap1 are also shown to assess the relative increase of the former with respect to the latter after addition of each nitrogen source. A Western blot from cells expressing the non-ubiquitinatable Gap1^K9R,K16R subjected to identical treatment is also shown as control to confirm that upper bands observed above the Gap1 band in the wild-type blots are ubiquitinated forms of the transceptor.

Fig 4

Non-metabolizable, transported and signalling amino acid analogues cause different effects for oligo-ubiquitination and endocytosis.A. Gap1-GFP localization in wild-type cells is shown 60, 120 and 180 min after addition of 5 mM of either the regular transported and signalling amino acid l-asparagine or the non-metabolizable, transported and signalling amino acid analogues β-alanine or d-histidine to nitrogen-starved cells.B. Analysis of Gap1 ubiquitination status in nitrogen-starved cells expressing endogenous GAP1 and induced with 10 μM CuSO₄ for 30 min prior to addition of nitrogen source, for expression of myc-ubiquitin from the P_CUP1-myc-Ubi URA3 plasmid, pMRT7. P13 fractions were collected at different time points (0, 30, 60, 120 and 180 min) after addition of 5 mM l-asparagine, β-alanine or d-histidine to nitrogen-starved cells. Upper panels: Western blot with anti-Gap1 antibody. Bottom panels: Western blot with anti-Pma1 antibody as loading control. Luminescent arbitrary units (LAU) × 10⁻⁶ are shown as ratio between the Gap1 band and Pma1 band for each time point to assess relative disappearance of the Gap1 band, consistent with endocytosis. The ratios between di- or tri-ubiquitinated to non-ubiquitinated Gap1 are also shown to assess the relative increase of the former with respect to the latter after addition of each nitrogen source. A Western blot from cells expressing the non-ubiquitinatable Gap1^K9R,K16R subjected to identical treatment is also shown as control to confirm that upper bands observed above the Gap1 band in the wild-type blots are ubiquitinated forms of the transceptor.

Fig 5

The non-transported and non-signalling competitive inhibitor of Gap1-mediated transport, l-Asp-γ-l-Phe, cannot trigger endocytosis but triggers ubiquitination in the wild-type strain.A. Progressive intracellular accumulation of radioactively labelled l-Asp-γ-l-Phe after addition of 5 mM of this compound to nitrogen-starved cells. Strains: wild-type (black bars), gap1Δ (white bars) and opt1Δ dal5Δ ptr2Δ (grey bars). Error bars represent s.d. between biological repeats.B. Growth of 1/10 serial dilution spottings of nitrogen pre-starved cells of the strains wild-type, gap1Δ, opt1Δ dal5Δ ptr2Δ and opt1Δ dal5Δ ptr2Δ gap1Δ on plates of nitrogen starvation medium (NSM) without or supplemented with 1 mM of l-citrulline, or l-Asp-γ-l-Phe. The same cells spotted in complete supplemented medium (CSM) are shown as positive growth control. Growth of the same cells in NSM + 1 mM of the dipeptide Leu-Met-NH2 or the tripeptide l-Arg-Gly-Gly is shown as control of peptide use as nitrogen source due to peptide carrier uptake.C. Localization of wild-type Gap1-GFP expressed in the strains gap1Δ and opt1Δ dal5Δ ptr2Δ gap1Δ is shown before and 60, 120 and 180 min after addition of 5 mM l-Asp-γ-l-Phe. The same cells exposed to 2.5 mM l-aspartate plus 2.5 mM l-phenylalanine is shown as control that the dipeptide constituent amino acids are able to induce endocytosis.D. Analysis of Gap1 ubiquitination status in nitrogen-starved cells expressing endogenous GAP1 (from the wild-type or the triple deletion mutant opt1Δ dal5Δ ptr2Δ) and induced with 10 μM CuSO₄ for 30 min prior to addition of nitrogen source, for expression of myc-ubiquitin from the P_CUP1-myc-Ubi URA3 plasmid, pMRT7. P13 fractions were collected at different time points (0, 30, 60, 120 and 180 min) after addition of 5 mM l-Asp-γ-l-Phe to nitrogen-starved cells. Upper panels: Western blot with anti-Gap1 antibody. Bottom panels: Western blot with anti-Pma1 antibody as loading control. Luminescent arbitrary units (LAU) × 10⁻⁶ are shown as ratio between the Gap1 band and Pma1 band for each time point to assess the relative disappearance of the Gap1 band, consistent with endocytosis. The ratios between di- or tri-ubiquitinated to non-ubiquitinated Gap1 are also shown to assess the relative increase of the former with respect to the latter after addition of each nitrogen source.

Fig 6

Behaviour of nearly transport-inactive Gap1^Y395C in the presence of non-signalling amino acids l-histidine and l-lysine.A. Transport of 5 mM l-citrulline, l-histidine or l-lysine in wild-type (black bars) or gap1^Y395C (white bars) strains. Error bars represent s.d. between biological repeats.B. Gap1^Y395C-GFP localization is shown 0, 60, 120 and 180 min after addition of 5 mM l-citrulline, l-histidine or l-lysine to nitrogen-starved cells.C. Analysis of Gap1 ubiquitination status in nitrogen-starved gap1Δ cells expressing Gap1^Y395C (from YCpGap1^Y395C, URA3 plasmid) and induced with 10 μM CuSO₄ for 30 min prior to addition of nitrogen source, for expression of myc-ubiquitin from the P_CUP1-myc-Ubi HIS3 plasmid, pMRT39. P13 fractions were collected at different time points (0, 30, 60, 120 and 180 min) after addition of 5 mM l-citrulline, l-histidine or l-lysine to nitrogen-starved cells. Upper panels: Western blot with anti-Gap1 antibody. Bottom panels: Western blot with anti-Pma1 antibody as loading control. Luminescent arbitrary units (LAU) × 10⁻⁶ are shown as ratio between the Gap1 band and Pma1 band for each time point to assess the relative disappearance of the Gap1 band, consistent with endocytosis. The ratios between di- or tri-ubiquitinated to non-ubiquitinated Gap1 are also shown to assess the relative increase of the former with respect to the latter after addition of each nitrogen source.

Fig 7

Gap1 transport activity at the plasma membrane causes signalling- and endocytosis-independent cross-endocytosis of transport-defective Gap1. Nitrogen-starved cells of strains coexpressing genomic mRFP-tagged wild-type or Gap1^K9R,K16R, combined with plasmid-expressed GFP-tagged wild-type or nearly inactive Gap1 (Y395C), were monitored (A) for mRFP and GFP localization at 0 (NSM) and 60 min after addition of 5 mM (B) l-citrulline, (C) l-histidine or (D) l-lysine.